Conformational comparisons of Pasteurella multocida types B and E and structurally related capsular polysaccharides

Abstract Pasteurella multocida, an encapsulated gram-negative bacterium, is a significant veterinary pathogen. The P. multocida is classified into 5 serogroups (A, B, D, E, and F) based on the bacterial capsular polysaccharide (CPS), which is important for virulence. Serogroups B and E are the primary causative agents of bovine hemorrhagic septicemia that is associated with significant yearly losses of livestock worldwide, primarily in low- and middle-income countries. The P. multocida disease is currently managed by whole-cell vaccination, albeit with limited efficacy. CPS is an attractive antigen target for an improved vaccine: CPS-based vaccines have proven highly effective against human bacterial diseases and could provide longer-term protection against P. multocida. The recently elucidated CPS repeat units of serogroups B and E both comprise a N-acetyl-β-D-mannosaminuronic acid/N-acetyl-β-D-glucosamine disaccharide backbone with β-D-fructofuranose (Fruf) side chain, but differ in their glycosidic linkages, and a glycine (Gly) side chain in serogroup B. Interestingly, the Haemophilus influenzae types e and d CPS have the same backbone residues. Here, comparative modeling of P. multocida serogroups B and E and H. influenzae types e and d CPS identifies a significant impact of small structural differences on both the chain conformation and the exposed potential antibody-binding epitopes (Ep). Further, Fruf and/or Gly side chains shield the immunogenic amino-sugar CPS backbone—a possible common strategy for immune evasion in both P. multocida and H. influenzae. As the lack of common epitopes suggests limited potential for cross-reactivity, a bivalent CPS-based vaccine may be necessary to provide adequate protection against P. multocida types B and E.


Introduction
Pasteurella multocida is a widespread pathogenic encapsulated gram-negative bacterium that primarily infects animals (Harper et al. 2006;Wilkie et al. 2012).The P. multocida causes a range of diseases in different host species: from fowl cholera in poultry, through hemorrhagic septicemia in cattle and buffaloes, atrophic rhinitis in swine, to snuffles in rabbits.Further, exposure to animals (e.g. through licks, bites, and scratches) causes occasional severe zoonotic infections with P. multocida in humans (Wilson and Ho 2013).Bovine hemorrhagic septicemia disease due to P. multocida is of particular concern due to the significant economic losses incurred worldwide, especially in low-and middle-income countries in Asia and sub-Saharan Africa where veterinary care is limited (Benikrane and De Alwis 2002).As antibiotics are only effective if started in the very early stages of diseasea challenge in environments with limited resources (Benikrane and De Alwis 2002;Lubroth et al. 2007)-hemorrhagic septicemia is primarily managed through relatively inexpensive whole-cell killed veterinary vaccines.However, these provide limited short-term protection of 6-12 months (Lubroth et al. 2007;Wilson and Ho 2013;Capik et al. 2021); more effective vaccines against P. multocida could therefore have significant benefit.
The P. multocida is classified into 5 serogroups (A, B, D, E, and F) based on the capsular polysaccharide (CPS) and 16 serovars based on cell wall lipopolysaccharide (Heddleston et al. 1972;Carter 1984).The capsular serogroups largely determine the hosts and diseases, with serogroups B and E associated with bovine hemorrhagic septicemia, serogroups A and F associated with fowl cholera, and serogroups D and A associated with porcine atrophic rhinitis (Wilson and Ho 2013).The CPS is an attractive vaccine antigen target, as it is essential for virulence of P. multocida and inhibits phagocytic uptake (Boyce and Adler 2000;Aktories et al. 2012).
Interestingly, PmE and PmB are structurally similar to the Haemophilus influenzae type e (Hie, Fig. 1C) and d (Hid,

Organism
Antigen RU structure in CASPER format a X represents a variable amino acid moiety: either L-alanine, L-serine, or L-threonine in the proportions 2:2:1.Fig. 1D) CPS, a possible example of convergent evolution.The H. influenzae is a leading cause of meningitis, otitis media among other infections in children (Richardson, Kuttel, et al. 2021).Type b was the primary cause of disease prior to the development of a vaccine; however, we are now seeing an increase in nonvaccine disease, especially due to type a with Hie and H. influenzae type f also showing increases (Slack 2021).Hie is very similar to PmB, lacking only the Gly substituent (Branefors-Helander et al. 1981a;Tsui et al. 1981), while Hid has a different backbone glycosidic linkage pattern (3-linked ManNAcA and 4-linked GlcNAc), lacks a Fruf substituent, and has a variable N-linked amino acid substitution pattern on C6 of ManNAcA (L-alanine, L-serine, Lthreonine in the proportion 2:2:1) (Branefors-Helander et al. 1981b;Tsui et al. 1981).
An understanding of the potential for cross-reactivity (and hence cross-protection) between related target antigenswhich is believed to have both structural and conformational aspects-may assist in the design of a minimal valency vaccine that provides maximum disease coverage (Kuttel and Ravenscroft 2018).The structural similarity between PmE and PmB points to the potential for cross-reactivity between these antigens, which would allow for a cheaper monovalent bovine vaccine.Early work using purified CPS in cattle challenge and passive mouse protection studies supports low levels of heterologous cross-reactivity and protection with good homologous serotype protection (Penn andNagy 1974, 1976;Nagy and Penn 1976).A more recent study, in which the CPS structures were elucidated for the first time, showed cross-reactivity between a PmB conjugate vaccine and PmE CPS, with limited cross-protection against PmE bacteria in mice (St Michael et al. 2021).The reciprocal study, however, was not performed and further work is still underway to extend these data (St Michael et al. 2021).In the absence of more detailed experimental information on crossreactivity, molecular modeling can provide potentially useful information on which epitopes are exposed and are therefore likely to be recognized by antibodies (Richardson, Kuttel, et al. 2021;Richardson et al. 2022).While not a current vaccine target, modeling the structurally related Hie and Hid CPS provides insight into the conformational effect of structural differences.
Here, we report a comparative modeling study to assess the conformational impact of the different structural features in the similar PmE, PmB, Hie, and Hid CPS antigens.We first compare the unsubstituted CPS backbones (PmE Fruf , PmB Fruf Gly, and Hid X-where X is the variable amino acid described above) to determine the effect of changes in the glycosidic linkages on the chain conformation (Fig. 2, top panel).Next, we investigate the impact of Fruf side chains on the backbone conformation (PmE and PmB Gly [Hie], Fig. 2, middle panel).Finally, the effect of the Gly amino acid side chain was established for PmB (Fig. 2, bottom panel).

Materials and methods
We followed our established systematic approach to the modeling of polysaccharides as previously described (Richardson, Kuttel, et al. 2021;Richardson, Ravenscroft, et al. 2021;Richardson et al. 2022).For each of the 6 molecules of interest (Fig. 1), we built CPS chains of 3 RU and 6 RU.
Chain length is an important consideration when modeling CPSs, as a short chain may have insufficient molecular flexibility, while long chains are more computationally expensive to model.On the basis of our previous work, we consider a 6 RU chain to be representative of the behavior of the longer polysaccharide.Further, antibodies bind small fragments of the CPS between 1 and 7 residues in length (Kabat 1966) corresponding to between 1 and 4 RU in the case of P. multocida, making a 6 RU model sufficient to explore antibody Ep.We perform 3 RU simulations to ensure the behaviors of the short 3 RU and longer 6 RU chains are consistent, but here, we present only the 6 RU results (3 RU results are available in the supplementary information).
Molecular dynamics simulations in the aqueous solution for each of the chains followed a protocol of initial system equilibration (200 ns) and then a production run of 800-1,800 ns (3 RU simulations) or 1,300-2,300 ns (6 RU simulations), respectively, as required for convergence.Data analyses were performed on these simulation trajectories as described below.

Molecular dynamics
The 3 RU and 6 RU chains were built using our CarbBuilder software (version 1.2.42) (Kuttel et al. 2011;Kuttel et al. 2016).Starting structures for each molecule were built using low-energy glycosidic linkage orientations obtained from potential mean force calculations (Supplementary Fig. S6) as per our previous work (Richardson, Ravenscroft, et al. 2021) The psfgen tool was used to create protein structure files (PSF) for simulation with the CHARMM36 additive force field for carbohydrates (Guvench et al. 2008(Guvench et al. , 2009)).For the PmB molecule with Gly, we used the CHARMM36 protein force field parameters for Gly, which are compatible with the CHARMM36 additive force field (Guvench et al. 2011).We modeled only the 6 RU model for this molecule.Starting structures were minimized with the Nanoscale Molecular Dynamics (NAMD) program (version 2.13) for 10,000 steps at 300 K. Minimized structures were solvated using the Visual Molecular Dynamics (VMD) software (Humphrey et al. 1996) solvation and ionization tools to add cubic TIP3P (Jorgensen et al. 1983) water boxes of 50 Å per side for the 3 RU systems and 70 Å per side for the 6 RU systems.Systems were then neutralized with 1 sodium (Na + ) counter ion per RU (3 ions for the 3 RU, 6 for the 6 RU).Initial minimization and heating protocols comprised 5 K incremental temperature reassignments beginning at 10 K up to 300 K with 500 steps of NAMD minimization and 8,000 steps of MD at each temperature reassignment.Solvated and ionized structures (Protein Data Bank and PSF files) for each 6 RU system are available as supplementary information.
Simulations of 3 RU and 6 RU were run using NAMD (version 2.13) (Phillips et al. 2005) with CUDA extensions for graphics processor acceleration (Stone et al. 2007).
Periodic boundary conditions equivalent to the cubic box size were employed for the solvated simulation with wrapping on.Long-range electrostatics were implemented with the Particle Mesh Ewald summation grid spacing set to 1 (Darden et al. 1993).Atoms were not held fixed, and the initial center of mass motion was turned off.The 1-3 pairs were excluded from nonbonded interactions, 1-4 interactions were not scaled, and the dielectric constant was set to 1. Smoothing functions were applied to both the electrostatics and van der Waal's (VDW's) forces with switching and cutoff distances of 10 Å and 12 Å, respectively.
A Leap-Frog Verlet integrator was used to integrate the equations of motion over a step size of 1 fs.A distance of 15 Å was used as the cutoff for inclusion in the pair list for the calculation of nonbonded forces.The short-range nonbonded interactions were calculated every 1 fs, full electrostatics calculations were performed every 2 fs, and atoms were reassigned every 10 fs (Van Gunsteren and Berendsen 1988).
Simulations were sampled under the isothermal-isobaric (nPT) ensemble.Langevin dynamics (Feller et al. 1995) were used to control the temperature with a damping coefficient of 5/ps.Nosé-Hoover Langevin piston dynamics were used as a barostat to maintain a target pressure of 1 atm (Nosé and Klein 1983;Hoover 1985).Variable system volume was used with a piston period of 100 fs and a decay of 50 fs.Simulations of 1,500 ns (2,500 ns for PmB Gly and PmB) were performed for the 6 RU systems comprising 200 ns of equilibration and 1,300 ns (2,300 ns for PmB Gly and PmB) of production run as was required for convergence (Supplementary Fig. S7).The 3 RU systems were run to 1,000 ns, except for PmE Fruf which was run to 2,000 ns, as required for convergence (data available as Supplementary Figs.S1  and S8).

Convergence
We addressed convergence using block standard averaging (Grossfield and Zuckerman 2009) applied to 2 metrics: end-to-end distance and radius of gyration (Supplementary Fig. S7).Block standard averaging was implemented with inhouse Python scripts.

Data analysis
Molecular conformations were visualized using VMD, with the PaperChain and Twister visualization algorithms used to highlight carbohydrate rings and chains (Cross et al. 2009), as required.
Trajectories were extracted at 25-ps intervals with analysis performed on frames 250 ps apart.Metrics, such as end-toend distances, were extracted from the simulation trajectories using Tcl scripting via VMD's Tk console.Data analyses were performed with in-house Python scripts and plots were generated using Matplotlib (Hunter 2007).

Chain flexibility
The end-to-end distance, r, was measured from C1 of Man-NAcA at the nonreducing end, to C3/4 of GlcNAc at the reducing end of each chain, thus excluding the highly flexible terminal residues.

Glycosidic linkages
Given the limited flexibility of the carbohydrate ring, the primary source of flexibility in the RU arises from the glycosidic linkages between neighboring monosaccharides.These glycosidic linkages are conveniently described by 2 dihedral angles, φand ψ.As per our previous work, we define φ = H 1 -C 1 -O 1 -C' x and ψ = C 1 -O 1 -C' x -H' x , with x representing the linkage position (Richardson, Ravenscroft, et al. 2021).In the case of the Fruf linkages which are (2 x)-linked, these definitions were adjusted to φ

Conformational analysis
The most common chain conformations for each simulation were determined by clustering the production trajectory frames into families and by calculating the relative occupancies of each family.Clusters comprising <6% of the production run (post equilibration) were discarded.Clustering was performed using the WMC PhysBio plug-in for VMD's builtin measure cluster command (Luis 2012).Prior to clustering, the molecules were aligned on the RU 3 and RU 4 residues excluding the Fruf /Gly residue(s) and any hydrogens on the backbone.Clustering was then performed as an RMSD fit to the ring and linkage atoms of the central 4 RUs of the chains excluding nonring atoms, the Fruf /Gly residue(s), and the highly flexible terminal RU 1 and RU 6.Five clusters were created with a cutoff of 3 Å.
Clustering analysis was also performed on the central 2 RUs, RU 3 and RU 4. Alignment was performed using the ManNAcA residue of RU 4 (excluding hydrogens) and was followed by clustering using the ring and linkage atoms of RU 3 and RU 4 excluding nonring atoms and Fruf /Gly residue(s), creating 3 clusters with a cutoff of 1.1 Å, before discarding clusters comprising <6% of the trajectory.

Solvent accessible surface
Hydrophilic/hydrophobic regions of the molecular surface were analyzed using VMD's built in "measure sasa" command.The solvent accessible surface area (sasa) analysis was performed by probing first hydrophilic regions (comprising hydroxyl groups, carbonyl groups, ring oxygens, nitrogen, and linkage oxygens) and then hydrophobic/neutral regions (comprising methyl groups, CH 2 groups, ring carbons, and ring protons) of the molecule using a VDW's radius of 2.5 Å-larger than that of water to imitate a potential small binding molecule.The ratio of hydrophilic sasa to total sasa was then calculated to determine the percentages of the hydrophilic and hydrophobic surface area exposed to solvent.

Results
We compare the effect of differing backbone linkages and substituents on the conformation and dynamics of the 6 CPS chains.We begin with a comparison of CPS chain extension and flexibility, followed by the molecular conformations, and finally analyze the potential binding epitopes (Ep) exposed on the saccharide chains.

Chain extension and flexibility
The molecular extension and flexibility of a carbohydrate is commonly measured by the fluctuation in the end-to-end distance, r, of the chain, during a simulation (Fig. 3A).Times series plots of r for the simulations of 6 RUs of each of the 6 CPSs reveal considerable differences in the chain extension and dynamics across the antigens (Fig. 3B-G, left column).This is consistent with the trend observed for the 3 RU chains (Supplementary Fig. S1).
The 3 unbranched CPS chains (Fig. 3B-D) have similar flexibilities (σ = 5 for all 3 6 RU molecules), with a wide range of conformations (see the trajectory snapshots at 50-ns intervals shown above the graphs).However, the backbones show considerably different chain extensions, as is clear from the r distribution histograms (Fig. 3, center column).PmE Fruf, comprising all β(1→3) glycosidic linkages, is the least extended chain, with a modal r value of 34 Å (Fig. 3B).Replacement of either of the 2 glycosidic linkages in the backbone with a β(1→4) linkage increases the length of the RU and thus the overall chain extension: Hid X (with a 4-linked GlcNAc) has an increased modal r value of 38 Å (Fig. 3D), and PmB Fruf Gly (with a 4-linked ManNAcA) is the most extended chain with a modal r value of 45 Å (Fig. 3C).
Addition of Fruf and/or Gly side chains to the ManNAcA backbone residue has little effect on the CPS chain extension, the modal r values remain very similar to the unsubstituted backbones: PmE 37 Å (Fig. 3E), PmB Gly 44 Å (Fig. 3F) and PmB 45 Å (Fig. 3G).Interestingly, side chains considerably decrease the flexibility of the β(1→3)-linked PmE (σ = 2), but not the β(1→4)-linked PmB molecules (σ = 5 for both).The decrease in PmE flexibility can be explained by the increased steric hindrance: The addition of a side chain to the already highly substituted ManNAcA residue (2-NAc, 5-COOH substituted, and 3-or 4-linked to neighbor) results in all ring hydroxyls being substituted.Close comparison of the r time series plots in Fig. 3C (PmB Fruf Gly) with Fig. 3F (PmB Gly) and G (PmB) shows a similar decreased flexibility of the chain on a ∼250-ns time scale.However, on a 1,000-ns time scale, new populations of very low r values appear, which maintain the overall chain flexibility on a level similar to the unsubstituted backbone.Interestingly, in the more substituted PmB, these populations occur more frequently than in PmB Gly.
The glycosidic linkages are the primary source of flexibility in the CPS chains.Side chains on ManNAcA reduce the overall flexibility of the chains through impeding rotation of the ManNAcA→GlcNAc glycosidic linkage through steric clashes between neighboring residues; rotation of the GlcNAc→ManNAcA is largely unaffected (Supplementary Fig. S2 and Supplementary Table S1).
In all cases, addition of the Fruf and/or Gly side chains reduces the flexibility of the CPS backbone.For PmE, a Fruf side chain increases the occupancy of the primary helical conformation from 51% (PmE Fruf , Fig. 4A) to 77% (PmE, Fig. 4B) and results in a more extended helix; the modal r distance increases from 34 Å to 37 Å (Fig. 3).Similarly, the 53% occupancy of the main helical conformation of PmB Fruf Gly (Fig. 4C) increases to 80% in PmB Gly (Fig. 4D), although in this case, the helices have similar extensions.The addition of a Gly side chain in PmB (Fig. 4E) does not alter the backbone conformation or flexibility of the primary helical conformation.Further, the Fruf (Fig. 4B, D, and E, magenta) or Gly (Fig. 4E, orange) side chains are highly and thus present potential targets for antibody binding while also shielding the backbone residues.The alignment of the hydrophobic (NAc, gray) and hydrophilic (COOH, red) residue substitutions differs in the 3 backbones, resulting in a considerable difference in surface hydrophilicity (Supplementary Fig. S5).For PmE Fruf (Fig. 5A), the NAc and COOH moieties are well separated on the backbone, whereas PmB Fruf Gly has frequent shielding of the groups of ManNAcA COOH by the NAc on an adjacent GlcNAc residue, resulting in a considerably less hydrophilic surface.However, the Fruf side chains with their many exposed hydroxyl groups increase the hydrophilicity of the molecular surface (Fig. 5, Quicksurf visualizations).This is particularly in the case of PmB (Supplementary Fig. S5), which has the potential to impact antibody binding.
As discussed above, the simulations of PmB Gly and PmB show minor populations with short r distances.
These populations are associated with bent conformations of the CPS chain.The bent conformations align adjacent NAc groups (Fig. 6A, E, and F), which may assist with conformational stabilization.

Epitopes
As the CPS regions bound by antibodies comprise 1-7 residues, it is useful to compare the 6 RU CPS chains on this length scale.Figure 6 focuses on the principal conformations of the 2 central repeat units (RU 3 and RU 4) for each of the 6 molecules; potential Ep are indicated by shaded boxes.There are key differences in the alignment of the side chains and residue substitutions, forming a range of different epitopes (despite a relatively conserved backbone).In the following  analysis, we focus on substitutions on opposing sides of the CPS chain, which we term as A and B.
For side A, the PmE Fruf molecule (Fig. 6A) has a hydrophobic epitope exposing 3 partially aligned N-acetyl groups, EpA-1.On side B, EpB-1 comprises the exposed COOH (red), of C6 on ManNAcA, and the N-acetyl on GlcNAc in the following RU.In PmE, the Fruf substitution considerably changes side A, forming the EpA-2 epitope with the 3 aligned NAc groups seen in EpA-1, now shielded by the Fruf residue.Similarly, on side B, the Fruf shields the exposed COOH forming the EpB-2 epitope (the NAc would also be shielded by the adjacent Fruf as in PmE the Fruf residues are on alternate faces).Conformationally, we see an increased conformational definition as the occupancy increases from 56% in PmE Fruf to 83% in PmE.
In PmB Fruf Gly (Fig. 6C), side A of the chain is considerably altered (compared to PmE Fruf ), with 1 NAc replaced by a hydrophilic carboxylic acid group and a further NAc joining the group, forming EpA-3.Side B remains similar to the PmE molecules, though, with the ManANAcA Nacetyl replacing the GlcNAc N-Acetyl-forming EpB-1 .Fruf substitution in PmB Gly (Fig. 6E) completely disrupts the molecular surface presented on both sides of the CPS with shielding Fruf residues forming epitopes EpA-4 and EpB-3.Conformationally, we see the occupancy increase from 47% to 51%.Additional Gly substitution in PmB (Fig. 6F) replaces COOH groups with bulkier Gly and thus further alters both sides of the chain, forming epitopes EpA-5 and EpB-4.The addition of Gly further increases the occupancy to 70% and suggests that for this longer, less compact, helix, an additional side group is required to achieve adequate shielding.
Interestingly, Hid X (Fig. 6C) has altered both sides of the chain, forming EpA-6 and EpB-5.On side A, the NAc pairs are sandwiched by COOH groups on either side with 1 / 4 -1 / 2 turn between NAc and COOH groups; on the mostly unsubstituted side B, the NAc of the ManNAcA is positioned where the COOH is in PmE and PmB.The central paired NAc in EpA-6 may be similar enough to EPA-1 to allow for antibody crossreaction.

Discussion
This study of 6 structurally similar CPS provides further evidence that small structural differences in a carbohydrate chain can have a large impact on both molecular conformation and the epitopes exposed for antibody binding.Although P. multocida types E and B (PmE and PmB) as well as the related Hie and Hid CPS molecules all have well-defined helical molecular conformations, the presence of β(1→4) glycosidic linkages in the backbone changes both the handedness of the helix and increases the extension of the helices relative to chains with only β(1→3) linkages.Further, side chain substitution of the backbone with Fruf decreases the molecular flexibility, with a concomitant increase in conformational definition.This has the potential to increase the antigenicity of the CPS fragments, as a more conformationally defined molecule presents a better target for antibodies.However, side chain substitutions also considerably alter the Ep and potentially shield the ManNAcA-GlcNAc backbone from antibody binding with the longer, more accessible serotype B helices, requiring >1 side group to achieve the same level of backbone shielding as those of serotype E. The ManNAcA and GlcNAc amino sugars are expected to be immunogenic.The Man-NAcA residue is often found with other amino sugars and is a motif commonly expressed by pathogens (Adlam et al. 1984;Yoneyama et al. 1984;Acker and Kammerer 1990).Further, mannose-binding proteins found in the lungs and serum of Bovidae (such as surfactant proteins, mannose-binding proteins, and collectins) are highly specific for fucose, mannose, N-acetyl-D-mannosamine, N-acetyl-D-glucosamine, and glucose, playing an important role in immune activation and bacterial clearance (Kawasaki et al. 1985;Holmskov et al. 1993;Neth et al. 2000).Therefore, we expect that the highly exposed Fruf and Gly substituents may be used to disguise an immunogenic backbone.
Our simulations suggest very limited cross-reactivity between any of the 6 CPSs we modeled, which is in line with the immunological data currently available.Early studies using whole-cell killed vaccines as well as CPS extracts demonstrated limited cross-protection between serotypes B and E (Penn andNagy 1974, 1976;Nagy and Penn 1976).Elucidation of the CPS structures containing the acidlabile Fruf (St Michael et al. 2021) allows interpretation of these experiments as well as the systematic modeling studies presented here.The significant differences that we have found in the CPS backbone conformations and lack of common epitopes between the CPS predict limited cross-reactivity and cross-protection of these 2 serotypes, therefore supporting the development of a bivalent CPS-based vaccine.A bivalent vaccine has been found to be effective against a type E challenge in cattle with passive mice protection against both types E and B, suggesting the cattle should also be protected against type B (Nagy and Penn 1976).
For the closely related Hie and Hid CPSs, we also suggest a lack of cross-reactivity as the Hid X CPS has very different conformations and potential Ep to the Hie (PmB Gly) and, most notably, the absence of the Fruf residue in Hid has an impact on the binding surface of the molecule as well as on the potential epitopes.This may also account for Hid being a less successful pathogen: The lack of Fruf side chain results in more exposure of the immunogenic backbone.Low levels of cross-reactivity between Hie and Hid may occur with limited cross-protection.Although the variable amino acid substituents of Hid (L-alanine, L-serine, and Lthreonine) were not modeled here, we expect a similar result with the amino acids having little impact on conformation but shielding the backbone although potentially less effectively than Fruf .
Our modeling investigation of Streptococcus pneumoniae serogroup 10 found that strongly cross-reactive serotypes share common epitopes (Richardson et al. 2022).Here, we find no evidence for common epitopes between the 6 CPSs studied, which suggest little potential for cross-protection between them.This is supported by early immunological studies with PmE and PmB CPSs that showed low levels of limited cross-protection.However, direct structural information on the interactions of CPSs with antibodies is required for corroboration of our prediction that the lack of crossprotection between the CPS is due to an absence of shared epitopes.Unfortunately, molecular binding studies of CPSs with bactericidal antibodies are not currently available and are rarely feasible.In the absence of direct structural data, immunological studies using reciprocal PmB and PmE CPS (or, preferably, the CPS conjugate vaccines and structural mutants modeled here) would assist in determining the importance of specific structural moieties/epitopes on the CPS for crossprotection.Such validation would be extremely valuable, as elucidation of the structural basis for cross-protection would greatly aid in the rational development of more effective CPSbased vaccines against P. multocida.

Fig. 2 .
Fig. 2. CPS molecules of PmE and B (PmB) and Hid and Hie modeled in this work (SNFG symbol representation).The top panel shows the unsubstituted backbone molecules: PmE without Fruf (PmE Fruf ), PmB without Fruf and Gly (PmB Fruf Gly equivalent to Hie without Fruf ), and Hid without the variable amino acid substituent (L-alanine, L-serine, or L-threonine in the proportions 2:2:1) represented by X (Hid X ).The middle panel shows the modeled Fruf -substituted molecules: PmE wild type, PmB without Gly (PmB Gly; equivalent to Hie wild type).The bottom panel shows the wild-type PmB, with the Gly substituent (PmB).

Fig. 3 .
Fig. 3. Time series of the end-to-end distance, r, in the 6 RU CPS chains.A) r is measured (Å) from C3/C4 of GlcNAc at the reducing end to C1 of ManNAcA at the nonreducing end, thus excluding the highly flexible terminal residues.Time series plots (left column) and corresponding histograms (center column) for the simulation trajectories are shown for: B) PmE Fruf , C) PmB Fruf Gly, D) Hid X , E) PmE, F) PmB Gly, and G) PmB; the initial 200 ns (indicated by lighter shade) are considered initial equilibration and the remaining trajectory is the production run.X represents a variable amino acid moiety: L-alanine, L-serine, or L-threonine.Conformational snapshots at 50-ns intervals are shown above the time series plots; SNFG line structures are shown in the right column.The histograms are labeled with the standard deviations (σ ) and modal peak r value.

Fig. 4 .
Fig. 4. Primary conformational families determined by cluster analysis of RU 2 to RU 5 of the CPS for A) PmE Fruf , B) PmE, C) PmB Fruf Gly, D) PmB Gly, E) PmB, and F) Hid X .The handedness of the helix is indicated on the left of the figure by left-handed (LH) or right-handed (RH).X represents a variable amino acid moiety: L-alanine, L-serine, or L-threonine.Conformations are visualized with the VDW (left) and Quicksurf representations (right).VDW representations were colored as for SNFG symbols: ManNAcA, green; GlcNAc, blue; NAc, gray; COOH, red; Fruf , magenta; and Gly, orange.For the Quicksurf representation, hydrophobic and neutral atoms were colored red, and hydrophilic atoms were colored blue.

Fig. 5 .
Fig. 5.The main conformational families of A) PmB Gly and B) PmB with NAc pairing of the nonprimary clusters indicated in zoomed in boxes.The VDW's representation was used with colors as per SNFG: ManNAcA residues are shown in green, GlcNAc residues in blue, NAc groups in gray, COOH groups in red, Fruf in magenta, and Gly in orange.

Fig. 6 .
Fig. 6.Main conformations and associated percentages identified from clustering of RU 3 and RU 4 only with conformational epitopes (Ep) indicated by shaded boxes for: A) PmE Fruf , B) PmE, C) PmB Fruf Gly, D) PmB Gly, E) PmB, and F) Hid X .X represents a variable amino acid moiety: L-alanine, L-serine, or L-threonine.The VDW's representation was used with colors as per SNFG: ManNAcA residues are shown in green, GlcNAc residues in blue, NAc groups in gray, COOH groups in red, Fruf in magenta, and Gly in orange.

Table 1 .
Line structures of PmB and PmE as well as Hie and d (Hid) RU CPSs.(Side groups indicated in bold).